MEASUREMENT OF ANTICOAGULANT EFFECTS OF NEW ORAL ANTICOAGULANTS PRACTICAL ASPECTS
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1 MEASUREMENT OF ANTICOAGULANT EFFECTS OF NEW ORAL ANTICOAGULANTS PRACTICAL ASPECTS Désirée Coen Herak Department of Laboratory Diagnostics University Hospital Centre Zagreb
2 WARFARIN ERA Warfarin monopoly on oral anticoagulant therapy, as well as that of other vitamin K antagonists derived from coumarins is over. Eby C. Clin Chem 2013; 59:
3
4 Warfarin monopoly on oral anticoagulant therapy, as well as that of other vitamin K antagonists derived from coumarins is over. WARFARIN ERA Within a few years it is likely that NOACs (DOACs) will be the dominant chronic anticoagulation therapy. Eby C. IJLH 2013; 35: POSTWARFARIN ERA
5 1109 articles (Medline search using terms NOAC, DOAC, TSOAC, apixaban, dabigatran, rivaroxaban, edoxaban AND laboratory, monitoring, measurement or test) Favaloro E, Lippi G. Semin Thromb Hemost 2015; 41:
6 Laboratory testing in the postwarfarin era - what do we need to know?
7 BJH 2012; 159: J Throm Haemost 2014; 12:
8 Recommendation from the SSC (2013) on Control of Anticoagulation for measuring NOACs ASSAYS SEMIQUANTITATIVE for the estimation of the relative intensity of anticoagulation indicators of supratherapeutic, therapeutic or subtherapeutic anticoagulation status QUANTITATIVE for the quantitative measurement of NOAC concentrations in plasma Assays should not be used for the quantitative measurement of NOAC concentrations.
9 QUANTITATIVE ASSAYS DIRECT LC tandem mass spectrometry gold standard provide accurate measure of a wide range of NOAC concentrations in plasma samples (especially for low concentrations) INDIRECT specific coagulation assays based on methods that are closely related to their anticoagulant targets and mechanism of action standard calibration curve has to be generated with drug specific calibrators
10 SEMIQUANTITATIVE ASSAYS THROMBIN TIME (TT) TT is very sensitive to the presence of dabigatran - suitable as a screening test for the exclusion of supratherapeutic dabigatran concentrations in plasma samples. Dabigatran concentration (μg/l) TT (s) BC Thrombin 21,6 127,5 >150 >150 >150 TT (ratio) 1,17 6,89 >8,11 >8,11 >8,11 N TT indicator of very low dabigatran concentration or no presence.
11 DILUTE THROMBIN TIME (dtt) The strong sensitivity of TT towards dabigatran led to the development of a CALIBRATED dtt using dabigatran standards to calculate dabigatran concentrations. Plasma samples are prediluted 1/8 or 1/20 in normal plasma before performing the assay. There is a linear relationship between dtt and dabigatran concentration in a wide range of concemtrations (a little bit not sensitive for low concentrations <50 ug/l). QUANTITATIVE ASSAY high correlation of dtt with dabigatran plasma concentrations measured by LC-MS/MS (Douxfils 2014)
12 DILUTE THROMBIN TIME (dtt) Commercially available kit HEMOCLOT ASSAY (HTI) In-house method BC Thrombin
13 Anticoagulant effects of NOACs on PT and APTT The anticoagulant effect of each NOAC on PT and APTT has been demonstrated in a number of studies. In general a more pronounced influence has been observed for: thrombin inhibitors on APTT FXa inhibitors on PT PT and APTT expressed in s and ratios show a dosedependent prolongation (increase) with increasing NOAC concentrations. However, remarkable between-reagent variability has been observed.
14 PT and APTT reagent sensitivity to Dabigatran PT reagents Thromborel S Recombiplastine 2G STA Neoplastin Cl plus Innovin STA Neoplastine R APTT reagents Actin FSL Actin FS Synthasil STA Cephascreen STA PTT-A Pathromtin SL Least sensitive Most sensitive
15 PT reagent sensitivity RIVAROXABAN Innovin Thromborel S STA Neoplastin Cl plus Recombiplastine 2G STA Neoplastine R APIXABAN Innovin Thromborel S STA Neoplastine R STA Neoplastin Cl plus Recombiplastine 2G Least sensitive Most sensitive
16 APTT reagent sensitivity RIVAROXABAN Synthasil STA Cephascreen Actin FSL APTT-SP Actin FS Pathromtin SL APIXABAN Actin FS Actin FSL STA Cephascreen Synthasil APTT-SP Pathromtin SL Least sensitive Most sensitive
17 Determination of local PT and APTT reagents sensitivities to NOAC IN-VITRO using NOAC specific calibrators (commercially available from several suppliers) EX-VIVO using plasma samples from patients receiving a specific NOAC (NOAC concentrations determined with the appropriate quantitative assay)
18 In-vitro determination of local PT and APTT sensitivity to NOACs 1. PT and APTT performed in pooled normal plasma samples spiked with increasing NOAC concentrations corresponding to through, peak and supratherapeutic levels: Dabigatran concentrations 0, 30, 150, 382 and 720 μg/l (Neqas supplementary exercise, April 2012) Rivaroxaban concentrations 0, 29, 121, 346 and 734 μg/l Apixaban concentrations 0, 33, 164, 398 and 737 μg/l (Neqas supplementary exercise, May 2014) 2. PT and APTT performed using commercial Rivaroxaban calibrators (STA-Rivaroxaban Calibrator, Stago, France) with concentrations 0, 100, 254 and 494 μg/l
19 Local PT and APTT sensitivity to NOACs Neqas supplementary exercise, April 2012 Neqas supplementary exercise, May 2014
20 Ratio Local PT and APTT sensitivity to Rivaroxaban determined using Rivaroxaban calibrators 2,25 2,00 1,75 PT Innovin (ratio) PT Thromborel S (ratio) PT Sclavo (ratio) APTT Actin FS (ratio) APTT Actin FSL (ratio) APTT Dapttin (ratio) APTT Sclavo (ratio) 1,50 1,25 1,00 0,75 0, Rivaroxaban concentration (µg/l)
21 Ex-vivo effect of rivaroxaban PATIENTS: 21 patient with nonvalvular atrial fibrilation (creatinine clearence >50 ml/min) receiving 20 mg of Rivaroxaban once daily SAMPLES: For each patient blood samples were taken: before the next dose (through concentration) 3h after drug administration (peak concentration ) METHODS: PT (Innovin), APTT (Actin FS) and anti-xa assay (Berichrom Heparin calibrated with STA-Rivaroxaban Calibrators)
22 Real-world variability in rivaroxaban levels in patients with atrial fibrilation
23 Rivaroxaban concentration (μmol/l) Rivaroxaban concentration (μmol/l) Real-world variability in rivaroxaban levels in patients with atrial fibrilation 250, , ,3 200, , ,0 150,0 100,0 100,0 72,2 50,0 50,0 15,9 0,0 C through C peak 0,0
24 Attempts to adapt the INR system for specific NOAC INR rivaroxaban = (PT patient /PT normal ) ISIrivaroxaban
25 Measurement of NOACs in other media The NOACs can be assesesed in alternate (nonplasma) samples. urine measurement of dabigatran and rivaroxaban (qualitative assesment) (Harenberg J et al. Semin Thromb Hemost 2013; 39: Harenberg J et al. Thromb J 2013; 11: article 15.) serum - measurement of rivaroxaban and apixaban (Harenberg J et al. Eur J Clin Invest 2014; 44: Harenberg J et al. Semin Thromb Hemost 2014; 40: )
26 Perspectives (the future) Indirect quantitative assays seem to be unreliable for the measurement of low plasma concentrations there is a need to develop specific coagulation assays that are accurate for these low concentrations. separate calibration curves in the low range? There is a need for the development of a global coagulation test which is sensitive to all NOACs and for which a cut-off associated with bleeding or thrombotic can be provided. drvvt seems to be promising POCT including alternate samples such as urine and serum is under development providing additional oportunities when plasma samples are unavailable.
27 Conclusions Up to now no biological assay can be recommended for the assesment of all NOACs. Each coagulation laboratory should be familiar with the sensitivity of its own PT and APTT reagents to each NOAC. A normal thrombin time suggests very low (not clinically relevant) level of dabigatran. For apixaban both PT and APTT are insensitive and patients may have normal coagulation times despite therapeutic concentrations, and anti-xa assays should be used to determine plasma concentrations. As there are no therapeutic intervals set, threshold values of NOAC concentrations that might still be assumed as safe must be defined.
28 Postwarfarin era and laboratory responsabilities Coagulation laboratory experts have a major role in ensuring patients receive appropriate testing and accurate interpretation of results, and must protect patients from inaccurate test results due to NOAC interference by educating clinicians and providing interpretive comments warning of possible confounding of results due to the presence of NOACs. Eby C. IJLH 2013; 35: The laboratory needs to know the drug being used in order to perform appropriate testing and apply the appropriate calibration curve.
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